Disruption of Plasmodium falciparum chitinase markedly impairs parasite invasion of mosquito midgut.

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasite Plasmodium gallinaceum appear to secrete products of two chitinase genes, to date only one chitinase gene, PfCHT1, has been identified in the nearly completed Plasmodium falciparum strain 3D7 genome database. To test the hypothesis that the single identified chitinase of P. falciparum is necessary for ookinete invasion, the PfCHT1 gene was disrupted 39 bp upstream of the stop codon. PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts in Anopheles freeborni midguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that the PfCHT1 gene product is necessary for ookinetes to invade the mosquito midgut.

Micronemal transport of Plasmodium ookinete chitinases to the electron-dense area of the apical complex for extracellular secretion.

Plasmodium ookinetes secrete chitinases to penetrate the acellular, chitin-containing peritrophic matrix of the mosquito midgut en route to invasion of the epithelium. Chitinases are potentially targets that can be used to block malaria transmission. We demonstrate here that chitinases of Plasmodium falciparum and P. gallinaceum are concentrated at the apical end of ookinetes. The chitinase PgCHT1 of P. gallinaceum is present within ookinete micronemes and subsequently becomes localized in the electron-dense area of the apical complex. These observations suggest a pathway by which ookinetes secrete proteins extracellularly.

Plasmodium falciparum phosphoenolpyruvate carboxykinase is developmentally regulated in gametocytes.

Plasmodium species have the capacity to fix carbon dioxide during intracellular development. This process contributes to the pool of free amino acids and metabolites, which are the end products of glucose metabolism in the malaria parasite. A gene encoding phosphoenolpyruvate carboxykinase (PEPCK), an enzyme known to catalyze CO(2) fixation was identified in the genome of the human parasite Plasmodium falciparum by DNA microarray analysis experiments and was cloned and characterized. PfPEPCK is a 66.2 kDa, ATP-dependent enzyme which is closely related to PEPCK from plants and yeast but markedly different from the host enzyme human PEPCK. PfPEPCK transcript and active enzyme levels are upregulated in the transmissible and zygote stages of parasite development relative to the asexual blood stages. Elevated expression of PfPEPCK during the extracellular zygote phase of P. falciparum development within the microenvironment of the mosquito midgut may reflect a glucose-rare medium and suggests a possible switch in carbohydrate metabolism to a gluconeogenesis pathway.

Shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria.

Malaria infects over 200 million individuals and kills 2 million young children every year. Understanding the biology of malarial parasites will be facilitated by DNA microarray technology, which can track global changes in gene expression under different physiological conditions. However, genomes of Plasmodium sp. (and many other important pathogenic organisms) remain to be fully sequenced so, currently, it is not possible to construct gene-specific microarrays representing complete malarial genomes. In this study, 3648 random inserts from a Plasmodium falciparum mung bean nuclease genomic library were used to construct a shotgun DNA microarray. Through differential hybridization and sequencing of relevant clones, large differences in gene expression were identified between the blood stage trophozoite form of the malarial parasite and the sexual stage gametocyte form. The present study lengthens our list of stage-specific transcripts in malaria by at least an order of magnitude above all previous studies combined. The results offer an unprecedented number of leads for developing transmission blocking agents and for developing vaccines directed at blood stage antigens. A significant fraction of the stage-selective transcripts had no sequence homologues in the current genome data bases, thereby underscoring the importance of the shotgun approach. The malarial shotgun microarray will be useful for unravelling additional important aspects of malaria biology and the general approach may be applied to any organism, regardless of how much of its genome is sequenced.

Plasmodium falciparum: histidine-rich protein II is expressed during gametocyte development.

Both early gametocytes (I-II) and asexual trophozoite stages of Plasmodium falciparum digest hemoglobin and detoxify haem by polymerizing it into parasite pigment called hemozoin. The mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate transport of hemoglobin to the food vacuole and catalyze the polymerization in asexual stages. We describe the transcription of histidine-rich protein II in gametocytes by Northern blot analysis and the expression of the protein in these stages by immunoprecipitation and Western blotting. Localization of histidine-rich protein II within the gametocyte by immunofluorescence assay and immunoelectron microscopy clearly illustrated the presence of this molecule in the infected red cell cytosol in the early stages of gametocyte development and internalization in the later gametocyte as it matures. There is a strong correlation between the stage-specific trafficking of histidine-rich protein II in gametocytes and the susceptibility of early but not late gametocytes to the antimalarial drug chloroquine.

Malaria transmission and naturally acquired immunity to PfEMP-1.

Why there are so few gametocytes (the transmission stage of malaria) in the blood of humans infected with Plasmodium spp. is intriguing. This may be due either to reproductive restraint by the parasite or to unidentified gametocyte-specific immune-mediated clearance mechanisms. We propose another mechanism, a cross-stage immunity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). This molecule is expressed on the surface of the erythrocyte infected with either trophozoite or early gametocyte parasites. Immunoglobulin G antibodies to PfEMP-1, expressed on both life cycle stages, were measured in residents from an area where malaria is endemic, Papua New Guinea. Anti-PfEMP-1 prevalence increased with age, mirroring the decline in both the prevalence and the density of asexual and transmission stages in erythrocytes. These data led us to propose that immunity to PfEMP-1 may influence malaria transmission by regulation of the production of gametocytes. This regulation may be achieved in two ways: (i) by controlling asexual proliferation and density and (ii) by affecting gametocyte maturation.

Virulence and transmission success of the malarial parasite Plasmodium falciparum.

Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.

The biology of Plasmodium falciparum transmission stages.

The most important function of any parasite is to secure transmission to new hosts. The gametocyte, the stage which has become developmentally committed to the sexual cycle, provides a critical link in the transmission of Plasmodium falciparum from the human host to the anopheline mosquito vector. It is therefore imperative that our determination to understand the biology of the gametocyte is greater than the technical obstacles which have resulted in the gametocyte being left very much out of the limelight by the intensive investigation of the asexual bloodstream parasite. Here we explore the areas of gametocyte biology which by nature of their relevance to control and pathology as well as basic biology, are the subjects of investigation in our laboratory. We also point out areas in need of particular attention.

CD36-dependent adhesion and knob expression of the transmission stages of Plasmodium falciparum is stage specific.

Plasmodium falciparum trophozoites sequester from the peripheral circulation by adherence to host endothelium. Gametocytes, also sequester during maturation. Analysis of the adhesion phenotype of stage I to V gametocytes of several isolates/clones was assessed by binding of infected cells to C32 melanoma cells (C32MC) and the purified adhesion proteins, leucocyte differentiation antigen (CD36) and intercellular adhesion molecule-1 (ICAM-1). These cells and proteins, have previously been shown to be receptors for adherence of trophozoites. Early gametocytes (stages I-IIA) were found to bind to C32MC as well as the purified receptor CD36 but not to ICAM-1. Early gametocytes bound to C32MC via CD36 and the parasite ligand involved in this binding was trypsin sensitive. Stage IIB to V gametocytes did not adhere to C32MC, CD36 nor ICAM-1. Electron-dense protruberances known as knobs and histidine rich protein 1 (HRP 1) expression have been associated with trophozite adhesion to CD36. Knobs were present at the surface of early but not late gametocyte infected cells. Stage-specific patterns of HRP 1 expression, consistent with a role for this molecule in CD36 adhesion of early gametocytes, were also observed. The adhesion phenotype of these young gametocytes was indistinguishable from that of the trophozoites by all criteria examined. These data support the hypothesis that other host receptors mediate the binding of late gametocytes.